Fusion protein having enhanced in vivo erythropoietin activity

ABSTRACT

Provided is a fusion protein comprising, at its carboxy terminal of human erythropoietin (EPO), a mutant having one to four amino acid substitutions in the carboxy terminal peptide (CTP) fragment of a human chorionic gonadotropin (HCG) β subunit, for increasing an in vivo half-life activity of EPO. The in vivo half-life can be greatly elongated while retaining the intrinsic activity of the EPO, without increasing the sugar chain content.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a fusion protein having anenhanced in vivo activity of an anti-pernicious anemia drug,erythropoietin (hereinafter, it is also referred to as “EPO”). Moreparticularly, the present invention relates to a fusion protein havingan enhanced EPO activity by increasing its in vivo half-life with itsown amino acid sequences, that is, without increasing the glycosylationcontent, wherein the fusion protein contains an EPO molecule fused to aparticular peptide naturally occurring in vivo.

[0003] 2. Description of the Related Art

[0004] EPO is a glycoprotein having a molecular weight in the range of30,000 to 34,000 Da, and is a hematopoietic factor promoting productionand differentiation of red blood cells. The glycoprotein binds to areceptor of precursor cells of red blood cells to initiate itshematopoietic activity and causes an increase in the amounts ofintracellular calcium ions, an enhancement of DNA biosynthesis andstimulation of hemoglobin formation. Also, recombinant human EPO(rhEPO)has been approved for the treatment of anemia associated with kidneyfailure, prematurity, hypothyroidism, malnutrition and so forth, andclinical use of rhEPO is continuously increasing. However, extensive useof rhEPO could be limited by inconvenience and high costs because rhEPOshould be administered about three times a week due to its shorthalf-life. Thus, the frequency of rhEPO administration for treatmentcould be reduced by maintaining an in vivo activity of EPO for a longertime.

[0005] In vivo biological activity of EPO is proportional to its in vivohalf-life which has been known to be related with the content of sialicacid located at the terminus of sugar chains in EPO. Thus, the in vivobiological activity of EPO is greatly dependent upon the presence orabsence of sugar chains. The types of sugar chains vary depending oncell types. Thus, when the same glycoprotein is expressed in differentcells, the types of sugar chains of the protein are characteristicallydifferent depending on the cell types. It is known that bacterial cells,for example, E. coli could not attach sugar chains to its proteins.Since it is known that proteins expressed in E. coli do not have anysugar chains, EPO expressed in E. coli does not contain sugar chains. Inthis case, EPO is confirmed to be biologically active in vitro but notactive at all in vivo. This is because EPO without sugar chains is morerapidly removed from the body, compared to EPO with sugar chains,resulting in an extremely short half-life. Consequently, the presence orabsence of sugar chains in EPO plays an important role in the biologicalactivity of EPO.

[0006] To date, a lot of researches have been vigorously carried out toincrease the biological activity of EPO. Most of these researches focuson substitution of some amino acids by inducing mutation of EPO genesusing mutagenesis techniques. For example, PCT/US94/09257 entitled“Erythropoietin Analogs”, filed by Amgen Inc., disclosed a method ofincreasing an in vivo half-life by increasing the sugar chain content inEPO through mutagenesis. Increasing an in vivo half-life through EPOdimer formation has been also attempted (A. J. Sytkowski et al., J.B.C.vol. 274, No. 35, pp 24773-24778). Other methods for increasing the invivo biological activity of EPO include fusing a novel amino acid,peptide or protein fragment to EPO molecules using genetic engineering,and to increase the sugar chain content in EPO, specifically the amountsof sialic acids. However, the kinds of amino acids, peptides or proteinfragments used in this method are very limited. In most cases, suchgenetic modifications may result in a decrease or loss in specificactivity of protein or cause antigenicity problems frequently occurringwhen those substances are used in vivo.

[0007] Researches into fusion proteins or chimeric proteins, rather thanEPO, have been carried out, and one of the examples thereof is afollicle stimulating hormone, which is a sex hormone (Furuhashi et al.,1995, Mol. Endocrinol). However, such proteins have not yet been appliedin the field because genetically modified proteins using geneticengineering pose several problems. It is not easy to obtain a modifiedtarget protein itself, requiring highly professional skills. Also, inmost cases, the activities of proteins may be undesirably decreased orremoved by addition of or substitution by new amino acids.

[0008] Under the circumstances, the present inventors began extensivestudies into the development of a new method of increasing the in vivoactivity of EPO by fusing new amino acids, peptides or proteins to EPOmolecules. In the course of carrying out these studies, it was foundthat a fusion protein obtained by fusing carboxy terminal peptide(hereinafter, it is also referred to as “CTP”) fragments of the βsubunit of a human chorionic gonadotropin (hereinafter, it is alsoreferred to as “HCG”) which is a protein naturally occurring in vivo, toEPO, dramatically increases the in vivo half-life of the EPO. Also, theEPO contains amino acids having the function of increasing glycosylationsites while retaining the intrinsic activity of the EPO (see KoreanPatent Application No. 10-2000-0075230).

SUMMARY OF THE INVENTION

[0009] The present inventors have unexpectedly discovered that CTPvariants whose glycosylation site in the peptide had been removed alsoremarkably increased the in vivo half-life of EPO. As a result of thisfinding, CTP variants which can increase the in vivo stability of EPOthrough use of amino acid sequences, without increasing the content ofsugar chains in EPO, have been developed, leading the present inventorsto complete the present invention.

[0010] The present invention, in which CTP variants whose glycosylationsites have been removed are used, is distinguishable from the prior art,in which an in vivo half-life is increased by increasing the content ofthe sugar chains in EPO, and is based on the discovery of CTP variantscapable of increasing the in vivo stability of EPO.

[0011] Accordingly, it is an objective of the present invention toprovide a fusion protein having an enhanced in vivo activity of humanEPO, wherein the fusion protein includes an EPO molecule fused to a CTPvariant of a HCG β subunit at its carboxy terminus.

[0012] It is another objective of the present invention to provide anucleic acid that encodes the fusion protein, a recombinant vectorcontaining the nucleic acid, and a cell line transfected with therecombinant plasmid.

[0013] It is still another objective of the present invention to providea method for preparing a fusion protein having an enhanced human EPOactivity by culturing the transformed cell line.

[0014] In one embodiment of the present invention, there is provided afusion protein having an enhanced in vivo activity of human EPO, whereinthe fusion protein includes an EPO molecule fused to a CTP variant of aHCG β subunit (hereinafter, it is also referred to as “ATP”) at itscarboxy terminus. Preferably, the CTP includes all or some of the aminoacids corresponding to positions 112-145, preferably to positions118-145, of the HCG β subunit, the amino acids being described by SEQ IDNo. 1.

[0015] ATP, which is a CTP variant, has the function of increasing thehalf-life of the EPO molecule with its own amino acid sequences, i.e,without increasing the sugar chain content of EPO.

[0016] Thus, unless the action of a target fusion protein increasing anin vivo EPO activity is adversely affected, positions and kinds of aminoacids experiencing a change in position within the above-noted range arenot specifically restricted. In other words, as long as the fusionprotein maintains the activity of increasing an in vivo EPO activity,amino acids at positions belonging to the above-noted range can bereplaced at any position, with any amino acids.

[0017] For example, it is preferable that ATP has one or more aminoacids substitutions at positions 121, 127, 132 and 138, and mostpreferably has serine (Ser) residues at positions 121, 127, 132 and 138replaced with alanine (Ala) residues (FIG. 1). In this case, the fusionprotein according to the present invention has the amino acid sequencedescribed by SEQ. ID No. 2. From the finding that Ser residues at theabove positions can be replaced with Ala residues, it is clear to oneskilled in the art that any other amino acid having similar size andcharge as Ala, for example, glycine (Gly), can replace Ser.

[0018] In another embodiment of the present invention, there is provideda nucleic acid that encodes the fusion protein, a recombinant vectorcontaining the nucleic acid, and a cell line transfected with therecombinant plasmid, preferably a Chinese hamster ovary (CHO) cell.

[0019] In still another embodiment of the present invention, there isprovided a method for preparing a fusion protein having an enhancedhuman EPO activity by culturing the transformed cell line.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above objects and advantages of the present invention willbecome more apparent by describing in detail preferred embodimentsthereof with reference to the attached drawings in which:

[0021]FIG. 1 depicts the base and amino acid sequences of ATP as a CTPvariant;

[0022]FIG. 2 depicts the base and amino acid sequences of a fusionprotein (EATP) of EPO and ATP;

[0023]FIG. 3 is a graphic representation illustrating production of anexpression vector pcDNA3.1-EATP;

[0024]FIG. 4 is an electrophoresis photograph of purified EATP; and

[0025]FIG. 5 graphically represents pharmacokinetic analysis results ofEATP and EPO.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will now be described in more detail. Thepresent invention is processed by steps of acquisition and cloning ofgenes of a target fusion protein, construction of expression vectors ofa target gene, transfection of animal cells and EATP expression, andpurification of the expressed EATP and activity measurement.

[0027] (1) Acquisition of Genes

[0028] Complementary DNA (cDNA) of EPO can be acquired by employing aconventional reverse transcription-polymerase chain reaction (RT-PCR)technique using a RT-PCT Premix Kit available from Bioneer Corp., Korea,in which primers EP1 and EC2 complementary to both terminals of EPO cDNApreviously prepared from a cDNA library of the human embryonic liver(available from Invitrogen Corp.) are used. EP1:ATGGGGGCACGAATGTCCTGCCTGGCTGG (SEQ ID NO:3) EC2: GTCCCCTGTCCTGCAGGCCT(SEQ ID NO:4)

[0029] EPO cDNA is cloned into a cloning vector pGEM-T (Promega Corp.),which is termed pGEMT-EPO, and its base sequence is identified for useas template in subsequent operations.

[0030] CTP variant genes of a HCG β subunit used in the presentinvention are obtained by artificial synthesis and self-priming PCR. Thesynthesized gene fragments are EA1, A2, A3 and A4: (SEQ ID NO:5) EA1:AGGGGAGGCCTGCAGGACAGGGGACTCCTCTTCCG (SEQ ID NO:6) A2: GGAAGGGCGGGGGGAGGGGCCTTG GC GGAAGAGGA (SEQ ID NO:7) A3: CCGC CCTTCCAAGCCCAGCCCGACTCCCGGGGCCG (SEQ ID NO:8) A4: TTATTGTGGGAGGATCGGGGTGTCG GCGGGCCCCG

[0031] (Bold Portions Indicate Portions for Amino Acid Replacement.)

[0032] Each 1 μL of four genes is taken (50 pmole/μL) to be subjected toPCR using a high fidelity Taq system (Boehringer Manheim Corp.).

[0033] Gene fragments (Modified CTP genes) of approximately 100 bps insize are identified in a 1% Agarose gel. These genes encode a peptideobtained by replacing 4 Ser residues at positions 121, 127, 132 and 138among 28 carboxy terminal amino acids at positions 118-145 of a HCG βsubunit, with Ala residues (see FIG. 1).

[0034] PCR is performed using a pGEMT-EPO as template and EP1 and EC2 asprimers, yielding only EPO genes. Then, PCR is further performed usingboth the EPO genes and the modified CTP genes as templates and usingEP11 and EP22 primers by means of the high fidelity Taq system, therebyacquiring a desired fusion protein with gene fragments of approximately630 bps (to be termed EATP genes). EP11: TAAGCTTATGGGGGTGCACGAATGT (SEQID NO:9) EP22: TGGATCCTTATTGTGGGAGGATCGGGGT (SEQ ID NO:10)

[0035] These genes are cloned into pGEM-T cloning vectors and then basesequences are identified (to be termed pGEMT-EATP) (see FIG. 3).

[0036] (2) Construction of Expression Vectors

[0037] pcDNA3.1 vector (Invitrogen Corp.) is used as an expressionvector. Both termini of the EATP gene in PGEMT-EATP have Hind III andBamH I restriction enzyme sites derived from the primers EP11 and EP22.pcDNA3.1 and the obtained pGEMT-EATP are treated with Hind III and BamHI. The linearized pcDNA3.1 and EATP gene are obtained from an Agarosegel using a Qiagen elution kit, followed by ligation, therebytransforming E. coli NM522. Plasmids are isolated from coloniesresulting after incubation overnight in an LB-Ampicillin solid medium,and are treated with the restriction enzymes Hind III and BamH I. Then,only colonies inserted EATP are selected by 1% Agarose gelelectrophoresis. The resultant plasmids are termed pcDNA3.1-EATP (seeFIG. 3).

[0038] (3) Transfection of CHO Cells and EATP Expression

[0039] CHO cells (DG44) are grown in a 60 mm dish to prepare 40-80%confluent cells (1-4×10⁵ cells/60 mm dish). 3 μL of a superfectionreagent (Boehringer Manheim Corp.) and 97 μL of media (α-MEM with media,serum free and non-antibiotic) are mixed sufficiently, and approximately2 μg of a plasmid pcDNA3.1-EATP DNA (more than 0.1 μg/μL) and 0.2 μg ofa dihydrofolate reductase (dhfr) gene containing vector pLTRdhfr26(ATCC37295) are added to the resultant mixture and reacted at roomtemperature for 5-10 minutes to then be added to the cells. After oneday, the media are replaced with α-MEM without media (containing 500 μg/mL G418) with 10% FBS. The cells are replenished with media with 500μg/mL G418 and cultured for 7-10 days. Then, cells withoutG418-resistant genes and cells of negative control group all die. Aftercells selected from the G418 media are sufficiently cultured, an EATPprotein expressed from the media is confirmed using an EPO ELISA kit(Boehringer Manheim Corp.).

[0040] (4) Purification of Expressed EATP

[0041] Using an anti-EPO monoclonal antibody (R&D Inc.), affinity resinsfor purification are prepared as follows.

[0042] 0.3 g of CNBr-activated Sepharose 4B is swollen in 1 mM HCl for20 minutes and loaded onto a column, followed by washing with 1 mM HCl.Then, the resultant resin is further washed in 4 mL coupling buffersolution (0.1 M NaHCO₃ and 0.5 M NaCl, pH 8.3) transferred to a tube andimmediately mixed with anti-EPO monoclonal antibody in the couplingbuffer solution (500 μg/vial), and then reacted at room temperature for2 hours. At this time, the tube is sufficiently shaken. Then, theresultant product is replaced with a blocking buffer (0.2 M glycine, pH8.0) and reacted at room temperature for 2 hours with agitation. Theresultant resin is washed sequentially with a 6.5 mL coupling buffersolution, a 6.5 mL acetate buffer solution (0.1 M acetic acid, 0.5 MNaCl, pH 4) and a 6.5 mL coupling buffer solution. The prepared resin ispacked into a column and then subjected to purification as follows.

[0043] Cells are grown in a serum-free medium for one day and then onlythe medium is concentrated approximately 5 times using a ultrafiltrationfilter, for example, Centriprep (having a nominal molecular weightcutoff of 10,000) (Millipore Corp.). Then, the concentrated solutionsare loaded onto a column equilibrated with phosphate buffered saline(PBS) at a flow rate about 20 mL/hr and washed again with PBS. Thetarget proteins are eluted in an elution buffer solution (0.1 M glycine,pH 2.8) and then immediately titrated with 1 M Tris solution foradjustment to pH 7.5. The purity of the purified EATP is 97% or higheras verified by SDS-PAGE and silver staining (see FIG. 4).

[0044] (5) Activity Measurement by Bioassay Test and BiochemicalAnalysis

[0045] Biological activities of the expressed and appropriately purifiedEPO and EATP are measured by a bioassay using spleen cells of a mousetreated with phenylhydrazine. The result shows that the activity of EATPis higher than that of EPO, suggesting that the presence of addedcarboxy terminals in EATP does not inhibit the activity of EPO.

[0046] (6) Pharmacokinetic Test

[0047] In order to confirm whether the prepared candidate materialsactually have a longer in vivo half-life, pharmacokinetic tests areperformed on mice. Here, the candidate materials are intravenouslyadministered to four mice at dosages of 20 units for each mouse. Toevaluate the concentration profile in blood, blood is gathered from themice and the concentration in the gathered blood is measured using anEIA kit (Boehringer Manheim Corp.). The pharmacokinetic test performedon mice shows that candidate material EATP has a much longer half-lifethan the control material EPO (see FIG. 5).

[0048] The present invention is further illustrated in the followingexamples, which should not be misconstrued as limiting the scope of theinvention.

EXAMPLE 1 Acquisition of Genes

[0049] cDNA of EPO was acquired by employing a conventional RT-PCRtechnique using a RT-PCT Premix Kit (Bioneer Corp., Korea), in whichprimers EP1 and EC2 complementary to both terminals of EPO cDNApreviously prepared from a cDNA library of the human embryonic liver(Invitrogen Corp.) were used. 30 cycles of PCR reactions were performedin the conditions of 35 seconds at 55° C. (annealing), 40 seconds at 72°C., and 20 seconds at 94° C., yielding EPO cDNA. The obtained EPO cDNAwas cloned into a cloning vector PGEM-T (Promega Corp.). In other words,the product of PCR was eluted from 1% Agarose, ligated to pGEM-T,followed by transformation of E. coli NM522. After overnight incubationin an X-gal/IPTG smeared LB-Ampicillin solid medium, plasmid DNA wasisolated from white colonies and reacted with restriction enzymes Sac Iand Sac II to select colonies having EPO cDNA inserts therein. Theobtained vector was termed pGEMT-EPO and its base sequence wasidentified for use as a template in subsequent processes.

[0050] Modified CTP genes of a HCG β subunit were obtained by artificialsynthesis and self-priming PCR. The synthesized gene fragments were EA1,A2, A3 and A4.

[0051] Each 1 μL of four genes was taken (50 pmole/μL) to be subjectedto 15 cycles of PCR using a high fidelity Taq system (Boehringer ManheimCorp.) under conditions of 40 seconds at 55° C. (annealing), 40 secondsat 72° C. and 20 seconds at 94° C. Gene fragments of approximately 100bps in size were identified in a 1% Agarose gel (modified CTP genes).

[0052] These genes encode a peptide obtained by replacing 4 Ser residuesat positions 121, 127, 132 and 138 among 28 carboxy terminal amino acidsof a HCG β subunit, with Ala residues (FIG. 1).

[0053] PCR was performed using a pGEMT-EPO template and EP1 and EC2primers, yielding only EPO genes. Then, 30 cycles of PCR were furtherperformed using both the EPO genes and the modified CTP genes obtainedas templates and using EP11 and EP22 primers by means of the highfidelity Taq system under conditions of 42 seconds at 57° C.(annealing), 60 seconds at 72° C., and 20 seconds at 94° C. Thus,approximately 630 bps of fused gene fragments were obtained (to betermed EATP genes). These genes were cloned into PGEM-T using abovementioned method (to be termed pGEMT-EATP), and its sequences wereidentified.

EXAMPLE 2 Construction of Expression Vector pcDNA3.1-EATP

[0054] pcDNA3.1 vector (Invitrogen) was used as expression vector. Bothterminus of the EATP gene in pGEMT-EATP have Hind III and BamH Irestriction sites derived from the primers EP11 and EP22.

[0055] pcDNA3.1 and the obtained pGEMT-EATP were treated with therestriction enzymes Hind III and BamH I. The linearized pcDNA3.1 andEATP gene were obtained from an Agarose gel using a Qiagen elution kit,followed by ligation, thereby transforming E. coli NM522. Plasmids wereisolated from colonies resulting after incubating overnight in anLB-Ampicillin solid medium, and were treated with the restrictionenzymes Hind III and BamH I. Then, only colonies inserted EATP wereselected by 1% Agarose gel electrophoresis. The resultant plasmids weretermed pcDNA3.1-EATP (see FIG. 3).

EXAMPLE 3 Transfection of CHO Cells and EATP Expression

[0056] CHO cells (DG44) were grown in a 60 mm dish to prepare 40-80%confluent cells (1-4×10⁵ cells/60 mm dish). 3 μL of a superfectionreagent (Boehringer Manheim Corp.) and 97 μL of media (α-MEM with media,serum-free and non-antibiotic) were mixed sufficiently, andapproximately 2 μg of a plasmid pcDNA3.1-EATP DNA (more than 0.1 μg/μL)and 0.2 g of a dihydrofolate reductase (dhfr) gene containing vectorpLTRdhfr26 (ATCC37295) were added to the resultant mixture and reactedat room temperature for 5-10 minutes and then added to the cells. Afterone day elapsed, the media were replaced with α-MEM without media(containing 500 μg/mL G418) with 10% FBS. The cells were replenishedwith media containing 500 μg/mL G418, and cultured for 7-10 days. Then,cells without G418-resistant genes and cells of negative control groupall died. After cells selected from the G418 media were sufficientlycultured, EATP protein expressed from the media was confirmed using anEPO ELISA kit (Boehringer Manheim Corp.).

EXAMPLE 4 Purification of Expressed EATP

[0057] Using an anti-EPO monoclonal antibody (R&D Inc.), affinity resinsfor purification were prepared as follows.

[0058] 0.3 g of CNBr-activated Sepharose 4B was swollen in 1 mM HCl for20 minutes and loaded onto a column, followed by washing with 1 mM HCl.Then, the resultant resin was further washed in 4 mL coupling buffersolution (0.1 M NaHCO₃ and 0.5 M NaCl, pH 8.3), transferred to a tubeand immediately mixed with anti-EPO monoclonal antibody in the couplingbuffer solution (500 μg/vial), and then reacted at room temperature for2 hours with agitation. At this time, the tube was sufficiently shaken.Then, the resultant product was replaced with a blocking buffer (0.1 Mglycine, pH 8.0) and reacted at room temperature for 2 hours. Theresultant product was washed sequentially with a 6.5 mL coupling buffersolution, a 6.5 mL acetate buffer solution (0.1 M acetic acid, 0.5 MNaCl, pH 4) and a 6.5 mL coupling buffer solution. The prepared resinwas packed into a column and then subjected to purification as follows.

[0059] Cells were grown in a serum-free medium for one day and then onlythe medium was concentrated approximately 5 times using aultrafiltration filter of Centriprep (having a nominal molecular weightcutoff of 10,000) (Millipore Corp.). Then, the concentrated solutionswere loaded onto a column equilibrated with PBS at a flow rate about 20mL/hr and washed again with PBS. The target proteins were eluted in anelution buffer solution (0.1 M glycine, pH 2.8) and then immediatelytitrated with 1 M Tris solution for adjustment to pH 7.5. The purity ofthe purified EATP was 97% or higher as verified by SDS-PAGE and silverstaining (see FIG. 4).

EXAMPLE 5 Activity Measurement by Bioassay Test

[0060] Phenylhydrazine was administered to a mouse once a day for 2 daysat the dose of 60 mg/kg. After 3 days, an enlarged spleen was isolatedfrom the mouse and pulverized with a homogenizer to gain spleen cells.The spleen cells were diluted to a concentration of 6×10⁶ cells/mL andeach 100 μL of the diluted sample was transferred to a 96-well plate.Standard EPO (0-500 mU/mL) and the expressed EPO and EATP (each 100mU/mL) were added to the respective wells. Then, the plate was stored ina CO₂ incubator maintained at 37° C. for 22 hours. 50 μL ofdimethyl-³H-thymidine (20 μCi/mL) was added to each well. The resultantplate was further reacted for 2 hours, and then the sample solutions ofeach well were adsorbed to a glass filter (Nunc 1-73164). The filter waswashed three times with saline and the radioactivity of the filter wasmeasured using a beta (β) counter. The measurements showed that theactivity of EATP was substantially equal to or slightly higher than thatof EPO, suggesting that the presence of added carboxy terminals in EATPdoes not inhibit the activity of EPO.

EXAMPLE 6 Pharmacokinetic Test

[0061] In order to confirm whether the prepared candidate materialsactually have a longer in vivo half-life, pharmacokinetic tests wereperformed on mice. Here, the fusion protein purified by the methoddescribed in Example 5 was intravenously administered to four mice atdosages of 20 units for each mouse. To evaluate the concentrationprofile in blood, blood was gathered from the mice at regular timeintervals, that is, every 30 minutes at the beginning and every 2 hoursafter 2 hours. and the concentration in the gathered blood wasdetermined using an EIA kit (Boehringer Manheim.). The result of thepharmacokinetic test is shown in FIG. 5. As shown in FIG. 5, thecandidate material EATP had a much longer (more than 2.5 times longer)half-life than the control material EPO.

[0062] According to the present invention, the in vivo activity of EPOcan be enhanced by increasing the in vivo half-life while retaining theintrinsic activity of the EPO with its own amino acid, i.e., withoutincreasing the sugar chain content of EPO.

1 10 1 34 PRT Homo sapiens Peptide (1)..(34) Amino acids in thepositions of 112 to 145 of human chorionic gonadotropin(HCG) beta submit1 Asp Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser 1 5 1015 Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu 20 2530 Pro Gln 2 220 PRT Artificial Sequence Fusion prfotein (EATP) oferythropoietin (EPO) and a variant of carboxy terminal peptide (ATP) ofhuman chorionic gonadotropin (HCG) beta subunit 2 Met Gly Val His GluCys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu ProLeu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp SerArg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn IleThr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr ValPro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu ValGly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser GluAla Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 Gln ProTrp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125 LeuArg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu 130 135 140Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr GlyAsp 180 185 190 Ser Ser Ser Ala Lys Ala Pro Pro Pro Ala Leu Pro Ser ProAla Arg 195 200 205 Leu Pro Gly Pro Ala Asp Thr Pro Ile Leu Pro Gln 210215 220 3 29 DNA Artificial Sequence Primer EP1 having the nucleotidesequence complementary to the terminal sequence of EPO cDNA 3 atgggggcacgaatgtcctg cctggctgg 29 4 20 DNA Artificial Sequence Primer EC2 havingthe nucleotide sequence complementary to the terminal sequence of EPOcDNA 4 gtcccctgtc ctgcaggcct 20 5 35 DNA Artificial Sequence variant ofHCG beta subunit CTP gene fragment EA1 5 aggggaggcc tgcaggacaggggactcctc ttccg 35 6 35 DNA Artificial Sequence Variant of HCG betasubunit CTP gene fragment A2 6 ggaagggcgg ggggaggggc cttggcggaa gagga 357 34 DNA Artificial Sequence Variant of Hcg beta subunit CTP enefragment A2 7 ccgcccttca agcccagccc gactcccggg gccc 34 8 35 DNAArtificial Sequence Variant of HCG beta subunit CTP gene fragment A4 8ttattgtggg aggatcgggg tgtcggcggg ccccg 35 9 25 DNA Artificial SequencePrimer EP11 for PCR to obtain the desired fusion gene EATP 9 taagcttatgggggtgcacg aatgt 25 10 28 DNA Artificial Sequence Primer EP22 for PCR toobtain the desired fusion gene EATP 10 tggatcctta ttgtgggagg atcggggt 28

What is claimed is:
 1. A fusion protein comprising human erythropoietin(EPO) having a carboxy terminal and a mutant linked to the carboxyterminal, said mutant being a carboxy terminal peptide (CTP) fragment ofa human chorionic gonadotropin (HCG) β subunit with one to four aminoacid substitutions in the CTP fragment.
 2. The fusion protein accordingto claim 1, wherein the CTP fragment includes all or some of the aminoacids corresponding to positions 112-145 of the HCG β subunit, the aminoacids being described by SEQ ID NO:
 1. 3. The fusion protein accordingto claim 2, wherein the mutant has one or more amino acid substitutionsat positions 121, 127, 132 and 138 of the HCG β subunit.
 4. The fusionprotein according to claim 3, wherein the mutant has serine residues atpositions 121, 127, 132 and 138 of the HCG β subunit replaced withalanine or glycine.
 5. A nucleic acid encoding the fusion protein asclaimed in claim
 1. 6. A nucleic acid encoding the fusion protein asclaimed in claim
 2. 7. A nucleic acid encoding the fusion protein asclaimed in claim
 3. 8. A nucleic acid encoding the fusion protein asclaimed in claim
 4. 9. A method for preparing a fusion protein having anenhanced in vivo EPO activity comprising culturing a cell linetransfected with a recombinant vector containing the nucleic acid asclaimed in claim
 5. 10. A method for preparing a fusion protein havingan enhanced in vivo EPO activity comprising culturing a cell linetransfected with a recombinant vector containing the nucleic acid asclaimed in claim
 6. 11. A method for preparing a fusion protein havingan enhanced in vivo EPO activity comprising culturing a cell linetransfected with a recombinant vector containing the nucleic acid asclaimed in claim
 7. 12. A method for preparing a fusion protein havingan enhanced in vivo EPO activity comprising culturing a cell linetransfected with a recombinant vector containing the nucleic acid asclaimed in claim 8.